KR890000537B1 - Process for preparing 5'guanylic acid through fermentation - Google Patents

Abstract

No content.

Description

Method for preparing 5'-guanylic acid by microorganism

The present invention relates to a method for preparing 5'-guanylic acid by converting 5'-xanthyl acid to 5'-guanylic acid using xantylic acid aminase produced by a special mutant. 5'-guanylic acid is known as a nucleic acid-based seasoning having a taste, and a method of converting 5'-xanthyl acid into 5'-guanylic acid is to add 5'-xanthyl acid to the xanthyl acid aminase-producing cell culture. Methods (Japanese Patent Application No. 46-39069, Small 47-41557) A mixed culture method (Japanese Patent Application No. 46-39070) for simultaneously culturing a converting strain and a 5'-xanthyl acid producing strain is known. However, these methods conventionally had the following problems in actual industrial use.

One . 5'-Xanthyl acid can not be converted directly to 5'-guanylic acid from 5'-xanthyl acid culture medium, or 5'-xanthyl acid can be recovered from converted cell culture medium and added to 5'-xanthyl acid culture solution to 5'-guanylic acid. The production cost of 5'-guanylic acid is increased because of the method for preparing the same.

2 . In addition to 5'-guanylic acid, guanosine diphosphate and guanosine triphosphate were produced or decomposed into guanine or guanosine to produce a mixture of guanine-based materials.

In order to solve this problem, the present inventors have studied a method of culturing a conversion strain directly to 5'-xanthyl acid culture solution and converting it to 5'-guanylic acid, and the conventional xanthyl acid amanase producing strain is 5'-xanthyl acid. When the concentration of 5'-xanthyl acid in the fermentation broth is 25-30 g / l or more, the growth of the fungus is inhibited and the xanthyl acid aminase activity is lowered, resulting in lower conversion yield and generation of unnecessary guanine mixture.

Therefore, the inventors of the present invention investigated the inhibition of growth of the transformed strains in the high concentration of 5'-xanthyl acid, and found that 5'-xanthyl acid in the culture directly affected the growth of the bacteria.

Therefore, the present inventors have completed the present invention by separating 5'-xanthyl acid-resistant strains which are not inhibited in growth even in a high concentration of 5'-xanthyl acid containing medium.

A special variant strain of Brevibacterium ammonia genes MW-6648 (KCTC 8201P) of the present invention is mutated in Brevibacterium ammonia genes ATCC 6872, resulting in a loss of nucleotide resolution and special xanthyl acid aminase activity by decoinine resistance. The modified known strain was isolated from the enhanced known MW-1672 strain.

The mutation treatment and screening method for separating the special variant strain MW-6648 of the present invention is as follows. Cells incubated for 12 hours at 34 ° C. in MW-1672 strains were suspended in 2 ml of 0.05 M phosphate buffer (pH 7.0), followed by N-methyl-N'-nitro-N-nitroso at a concentration of 500 µg / ml. 2 ml of guanidine solution (dissolved in 0.05M phosphate buffer solution) was added and shaken at room temperature for 20 minutes, followed by centrifugation and washing to separate cells and mutant organic agents. It was. By taking the excellent bacterial colonies, shaking cultured at 34 ℃ for 24 hours in the medium of week 3, and measuring the bacterial growth concentration, the strains with superior bacterial growth compared to the comparative group (MW-1672 phagocytosis) were selected, and there was no nucleotide degrading ability and no xan The strain of the present invention was finally isolated in a medium having strong thymic acid aminase activity and good growth in a medium having a concentration of 5'-xanthyl acid 60 mg / ml.

Note 1) 20 g of glucose, 1 g of magnesium sulfate monohydrate, 50 mg of calcium chloride dihydrate, 10 mg of manganese sulfate monohydrate, 1 mg of zinc sulfate monohydrate, 10 mg of ferrous sulfate monohydrate, 1 g of ferric phosphate, 1 g of dibasic phosphate, 1 g of sulfuric acid 2 g ammonium, 3 g urea, 5 mg thiamine hydrochloride, 10 mg calcium pantothenate, 5 mg nicotinic acid, 430 µg biotin, 20 mg adenine, 20 mg guanine, 20 g agar, 1 l distilled water, pH 8.0 controlled 15 minutes sterilization at 120 ° C.

Note 2) Add 5 g of yeast extract and 60 g of 5'-xanthyl acid to the medium of Note 1.

Note 3) 5'-xanthyl acid fermentation broth (5'-xanthyl acid concentration 53g / l) 1 l (Production strain: Brevibacterium ammonia Genes MW-4768-6) 30 g of glucose, 1 g of magnesium sulfate heptahydrate, 5 g of corn extract , 4 g of phosphoric acid, 3 g of hydroxide, 5 g of ammonium sulfate, 30 µg of biotin, 20 mg of adenine, 10 mg of guanine, sterilized for 15 minutes at 115 ° C.

The difference between the mutant MW-6648 strain and the parent strain MW-1672 strain of the present invention is as follows.

TABLE 1

Comparison of Physiological Characteristics

Note 4) Nutritional requirements and sugar magnetization capacity were investigated by the exclusion and addition of the irradiated material based on the medium of Note 1, and xanthylamide aminase activity was reported in the Journal of Biochemistry Vol. 63, No. 63, No. 6, page 661. According to the method of the Journal of Biochemistry Vol 63 No 3. P 661) and the method of HS Moyed and B magasanik was expressed as an increase in OD for comparison at 295mμ.

TABLE 2

Growth Growth in 5'-Xanthyl Acid Addition Medium and 5'-Xanthyl Acid Fermentation Broth

Note 5) +++: growth and growth, ++: normal growth, ±: almost no growth,-: no growth The medium was incubated at 1 and 2, 34 ℃ for 72 hours to examine the growth.

TABLE 3

Comparison of Growth Rate by Nutrient Substances in Liquid Culture

Note 6) The control strain of the culture medium (except agar) of week 1 measured the absorbance at 610 ㎛ when the growth of each material was excluded from the medium of week 1. Culture conditions: 50ml shake in a 500ml flask cultured for 48 hours at 34 ℃.

As can be seen in the physiological comparison experiments of Tables 1, 2, and 3 above, the physiological properties of the special mutant strains of the present invention promote the growth of adenine / biotin essential and chiamine hydrochloride in nutritional needs of the parent strain MW-1672. The peculiarity of the mutant strain MW-6648 is that Mars magnetizes only fructose glucose, but its physiological and culture characteristics are clearly distinguished from those of known MW-1672 strains because of its nutritional requirement, which promotes the growth of biotin essential, adenine, guanine and calcium pantothenate. In particular, since it is resistant to 5'-xanthyl acid, it has high xanthyl acid aminase activity without being inhibited in growth even in the high concentration of 5'-xanthyl acid culture, so it can be directly converted into 5'-guanylic acid from 5'-xanthyl acid culture. It is judged to be an excellent variant suitable for the evolving 5'-guanylic acid production method.

As the medium of the strain of the present invention, hydrolyzate of glucose, fructose, mannose, and polysaccharides constituting them can be used as the carbon source, and ammonia and ammonia salt urea are used as the nitrogen source. According to the physiological properties of the bacteria, various inorganic and organic nutrients are added, and yeast extract, meat extract, corn steep liquor, peptone and the like are effective.

The mutant strain of the present invention is grown in an aqueous solution containing the above nutrients, that is, a conventional microbial medium, as well as a medium in which the above nutrients are added to a high concentration of 5'-xanthyl acid fermentation broth, and the xanthanate aminase is produced in the medium. '-Xanthyl acid is efficiently converted to 5'-guanylic acid. After the incubation, 5'-guanylic acid produced by a known ion exchange resin adsorption elution method is recovered.

A detailed method for producing 5'-guanylic acid using the mutant strain of the present invention is described in the following examples, but the present application is not limited to the examples.

Example 1

Use strain: special variant strain MW-6648 of the present invention

Species culture: Glucose 40g, 3g first phosphate, 3g second phosphate, 5g peptone, 5g corn leachate, 6g ammonium sulfate, 3g urea, 0.1g calcium chloride dihydrate, 10mg ferrous sulfate heptahydrate, 1 manganese sulfate Hydrate 10mg, Zinc Sulfate Heptahydrate 1mg, Biotin 50㎍, Nicotinic Acid 5mg, Pantothenate Calcium 10mg, Adenine 30mg, Guanine 30mg, Distilled Water 1l, 5N-KOH Autoclave for minutes. After the seedlings were inoculated with 1 platinum, shaking culture was performed at 32 ° C. for 24 hours to prepare a seed culture solution.

Production medium composition and culture method

5 l of 5'-xanthyl acid culture (containing 62 mg / ml of 5'-xanthyl acid) was heated and sterilized at 80-90 ° C, followed by the addition of 7 medium a week, and then inoculated with 300 ml of the seed culture cultured as described above. After incubation for 20 hours at pH 6.8-7.2, aeration 0.5-1VVM, 600rpm, the medium of week 8 is added and the culture is continued. At 35 hours after incubation, the incubation temperature is adjusted to 43-45 ° C., pH 7.4-7.6, aeration 0.5 VVM, and 9 times a week is added. Thereafter, the culture time was added twice so that the magnesium sulfate was 0.1-0.05% at intervals of 2-3 hours, and the fermentation was terminated at 43 hours from the start of the culture. The concentration of 5'-guanylic acid in the final fermentation solution was 48.5 mg / ml, and there were trace amounts of guanine-based mixture and 5'-xanthyl acid in addition to 5'-guanylic acid. 2 l of the culture medium from which the cells were removed was passed through a strong acid cation exchange resin and a strong base anion exchange resin, and 5'-guanylic acid was recovered and concentrated according to a conventional method to obtain 89.4 g of crude crystals.

Note 7) Sterilized for 20 minutes at 300 g of glucose, 8 g of phosphoric acid, 7 g of hydroxide hydroxide, 4 mg of magnesium sulfate heptahydrate, 10 mg of calcium pantothenate, 10 mg of nicotinic acid, 10 mg each of adenine and guanine, and 400 ml of distilled water for 20 minutes.

Note 8) 400 g of glucose is dissolved in distilled water to make 500 ml. Sterilize at 120 ° C. for 20 minutes.

Note 9) Dissolve 180 g of glucose, 20 g of trisodium phosphate 12 hydrate, 10 g of magnesium sulfate heptahydrate, and 10 g of alkyl dimethylbenzyl ammonium chloride in distilled water to make 300 ml. Sterilize at 120 ° C. for 20 minutes.

Note 10) N.B.S.14l was used for the fermenter, and HPLC was used for 5'-guanylic acid, 5'-xanthyl acid and guanine mixture analysis.

Note 11) 5'-xanthyl acid fermentation broth 5'-xanthyl acid production strain was cultured by a well-known method using the Btevi bacterium ammonia Genes MW-4768-8.

Example 2

It was cultured according to Example 1 using starch saccharification instead of glucose, and the concentration of 5'-guanylic acid of the final fermentation was 32.4 mg / ml. 5'-Xanthyl acid, guanosine triphosphate, guanosine diphosphate, guanine and guanosine other than 5'-guanylic acid were hardly detected.

Claims (1)

A method for producing 5'-guanylic acid by microorganisms, characterized by culturing a special variant MW-6648 (KCTC 3201P) of Brevi bacterium ammonia genes in a medium containing 5'-xanthyl acid.